Gestational diabetes (GDM), or hyperglycemia that first manifests during pregnancy, worsens pregnancy outcomes and long-term health risks for both a mother and her offspring. As with all types of diabetes, a relative insufficiency of functional pancreatic ?-cells is a fundamental defect contributing to GDM. Normally, ?-cells adapt to the metabolic challenges of pregnancy by expanding ?-cell mass. This expanded mass regresses in the postpartum period. Thus, pregnancy is a unique physiologic condition that occurs in a fully-developed adult and requires rapid, dynamic changes in ?-cell mass. Unfortunately, the mechanisms of normal gestational ?-cell adaptation and the defects underlying GDM are poorly understood. Our long-term goal is to understand the mechanisms regulating ?-cell proliferation and mass during pregnancy, in order to leverage that knowledge for therapeutic expansion of ?-cells in all types of diabetes. Building on our work establishing that loss of prolactin receptor (PRLR) signaling in ?-cells results in GDM, we recently identified novel PRLR differentially expressed genes (PRLR-DEGs) and key transcriptional regulators of PRLR-DEG expression. The objective of this grant is to precisely define how PRLR regulates ?-cell gene expression during pregnancy and the postpartum period. We propose the central hypothesis that PRLR signaling orchestrates an anticipatory transcriptional program of ?-cell mass expansion during gestation and survival of adequate ?-cell mass during postpartum regression. We will test this hypothesis with the following Specific Aims: (1) elucidate transcriptional mechanisms regulating PRLR-DEGs within ?-cells during pregnancy. To do so, we will use ChIP-seq and ATAC-seq to examine how PRLR-DEGs are regulated in mouse and human islets during pregnancy or in response to prolactin stimulation. In Aim (2) we will define PRLR signaling-dependent and -independent ?-cell subpopulations during pregnancy using single-cell RNA sequencing, lineage tracing and colocalization studies. For Aim (3) we will identify mechanisms of ?-cell survival during ?-cell mass regression in the early postpartum period through pulse-chase labeling and lineage tracing of ?-cells that proliferated during pregnancy, as well as examine how inducible loss of PRLR specifically within the postpartum period affects ?-cell mass. Together, results from these studies will reveal transcriptional mechanisms downstream of PRLR and illuminate unique aspects of gestational proliferation (Aim 1), define ?-cell subpopulations spatially and temporally across pregnancy (Aim 2), and establish a new role for PRLR signaling in regulation of ?-cells postpartum (Aim 3). Our research is significant because these findings would clarify mechanisms of gestational ?-cell adaptation and expand our understanding of how PRLR activation regulates transcription. At a fundamental level, these studies will expand our understanding of the mechanisms regulating dynamic changes in ?-cell mass, which may identify novel strategies to promote ?-cell expansion for therapeutic purposes.